USPTO Patents Application 10670724

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CELL TISSUE SYSTEMS

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PATENT APPLICATOR
IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
In re the Application of

Kelvin G.M. BROCKBANK et al. Group Art Unit: 1651

Application No.: 10/670,724 Examiner: S. SAUCIER

Filed: September 26, 2003 Docket No.: 113024

For: METHOD FOR TREATMENT OF CELLULAR MATERIALS WITH SUGARS
PRIOR TO PRESERVATION

DECLARATION UNDER 37 C.FJL 61.131
I, Kelvin G. M. BROCKBANK, a citizen of the United Kingdom, hereby declare and

state:

1 . This Declaration is submitted as evidence that the subject matter claimed in
the above-identified application was invented prior to July 26, 2002, the earliest U.S. filing
date of U.S. Patent Publication No. 2005/027710 ("Toner").

2. I am a named inventor in the above-captioned patent application.

3. I am one of the authors of the attached Invention Proposal ("IP"), dated prior to
July 26, 2002, a true copy of which appears as Exhibit A attached to this Declaration.

4. Exhibit A describes a method for preserving living cellular material,
comprising incubating the cellular material in a culture medium containing at least one sugar
for at least three hours; and after the incubation, subjecting the cellular material to a
preservation protocol, wherein the culture medium contains sugar, and wherein the at least
one sugar comprises trehalose. Exhibit A further describes a method for preparing living
cellular material for preservation, comprising incubating the cellular material in a culture
medium containing at least one polysaccharide for at least three hours, wherein the culture

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Application No. 10/670,724

medium contains polysaccharide, and wherein the at least one polysaccharide comprises
trehalose. In particular, the specifics of the method for preserving living cellular material
and a method for preparing living cellular material for preservation disclosed in the above-
identified application are described on pages 2 through 4 of Exhibit A.

5. The remainder of the IP sets forth further details of the design. For example,
details of the incubation time, sugars, preservation protocol, viability, etc., are described on
pages 2 through 4 of the IP.

6. The invention described in Exhibit A may thus be summarized as follows:

(a) a method for preserving living cellular material or a method of preparing
living cellular material, comprising incubating the cellular material in a culture medium
containing at least one sugar for at least three hours; and

(b) if preserving the living cellular material, after the incubation, subjecting the
cellular material to a preservation protocol,

(c) wherein the culture medium contains sugar, and

(d) wherein the at least one sugar comprises trehalose.

7. Exhibit A describes an invention conceived and reduced to practice prior to
July 26, 2002. This invention is claimed in the above-identified application.

8. Prior to July 26, 2002, r and/or those under my direct supervision and control,
carried out a reduction to practice of the invention described in Exhibit A and thereby
provided the methods described in paragraph 4-7 herein.

I hereby declare that all statements made herein of my own knowledge arc
true, and that all statements made on information and belief are believed to be true; and
further that these statements were made with the knowledge that willful false statements and
the like so made are punishable by fine and/or imprisonment under Section 1001 of Title 18

-2-

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Application No. 1 0/670,724
of the United States Code, and that such willful false statements may jeopardize the validity
of the application or any patent issuing therefrom.

Date: 1 3^ ^*>g(>

EXHIBIT A

Method for Treatment of Cellular Materials with Sugars
Prior to Preservation

Field of the invention: The invention relates to the field of cell, tissue and organ preservation.
More specifically, the invention relates to a method for treatment of cellular materials with
sugars prior to preservation that results in enhanced cell survival post-preservation. This is
particularly important because the sugars, trehalose and sucrose, are not cytotoxic to cells and
may not have to be removed before living, natural or man-made biological materials are
transplanted into humans or animals.

Background of the invention: Conventional approaches to cryopreservation that provided the
cornerstone of isolated cell storage have not been successfully extrapolated to more complex
natural, or engineered, multicellular tissues. Tissues are much more than simple aggregates of
various cell types; they have a highly organized, often complex, structure that influences their
response to freezing and thawing. The formation of extracellular ice, in particular, which is
generally regarded as innocuous for cells in suspension, is known to be a hazard to structured
tissues and organs. Cryopreservation is a complex process of coupled heat and mass transfer
generally executed under non-equilibrium conditions. Advances in the field were modest until
the cryoprotective properties of glycerol and dimethyl sulfoxide (DMSO) were discovered in the
mid 1900's. 1,2 Many other cryoprotective agents (CPAs) have since been identified.
Combinations of CPAs may result in additive or synergistic enhancement of cell survival by
minimization of intracellular ice during freezing. 3

Restriction of the amount and size of extracellular ice crystal formation during cryopreservation
can be achieved by using high concentrations of CPAs that promote amorphous solidification,
known as vitrification, rather than crystallization. 4 Vitrification is a relatively well understood
physical process, but its application to the preservation of biological systems has not been
without problems, since the high concentrations of CPAs necessary to facilitate vitrification are
potentially toxic. To limit toxic effects it is necessary to use the least toxic CPAs at the lowest
concentrations that will still permit glass formation (at cooling rates that are practical for bulky
mammalian tissues). 4 Comparison of the effects of vitrification and conventional frozen
cryopreservation upon venous contractility demonstrated that vitrification is superior to
conventional cryopreservation methods. 4 Vitrification has more recently been used effectively
for a variety of other tissues including myocardium, skin, and articular cartilage.

However, both conventional freezing and vitrification approaches to preservation have
limitations. First, both of these technologies require low temperature storage and transportation
conditions. Neither can be stored above their glass transition temperature for very long without
significant risk of product damage due to ice formation and growth. Both technologies require
competent technical support during the rewarming and CPA elution phase prior to product
utilization. This is possible in a high technology surgical operating theater but not in a doctor's
outpatient office or in third world environments. In contrast, theoretically, a dry product would

have none of these issues because it should be stable at room temperature and rehydration should
be feasible in a sterile packaging system.

Drying and vitrification have previously been combined for matrix preservation of
cardiovascular and skin tissues, but not for live cell preservation in tissues or engineered
products. Nature, however, has developed a wide variety of organisms and animals that tolerate
dehydration stress by a spectrum of physiological and genetic adaptation mechanisms. Among
these adaptive processes, the accumulation of large amounts of disaccharides, especially
trehalose and sucrose, is especially noteworthy in almost all anhydrobiotic organisms including
plant seeds, bacteria, insects, yeast, brine shrimp, fungi and their spores, cysts of certain
crustaceans, and some soil-dwelling animals. 5 " 7 The protective effects of trehalose and sucrose
may be classified under two general mechanisms: (1) "the water replacement hypothesis" or
stabilization of biological membranes and proteins by direct interaction of sugars with polar
residues through hydrogen bonding, and (2) stable glass formation (vitrification) by sugars in the
dry state.

The stabilizing effect of these sugars has also been shown in a number of model systems,
including liposomes, membranes, viral particles, and proteins during dry storage at ambient
temperatures. 8 " 10 On the other hand, the use of these sugars in mammalian cells has been
somewhat limited, mainly because mammalian cell membranes are believed to be impermeable
to disaccharides or larger sugars. 1 1 For sugars to be effective they need to be present both on the
inside and the outside of the cell membrane. Several methods have been developed for loading
of sugars in living cells. Recently, a novel, genetically-modified, metal-actuated switchable
membrane pore has been used to reversibly permeabilize mammalian cells to sugars with
significant post-cryopreservation and, to lesser extent, drying cell survival. 12 Other permeation
technologies, that have been considered for placing sugars in cells include use of pressure,
electroporation, microinjection, and thermal and osmotic shock. The expression of sucrose and
trehalose synthase genes and transporters has also been considered as means for delivery of
sugars into cells. Introduction of trehalose into human pancreatic islet cells during a cell
membrane thermotropic lipid-phase transition, prior to freezing and in the presence of a mixture
of 2M DMSO and trehalose, resulted in previously unattainable cell survival rates. This
method depends upon suspension of cells in a trehalose solution and either cooling or warming
the solution through the thermotropic transition of the cells. 14 Human fibroblast transfection with
E. coli genes expressing trehalose resulted in retention of viability after drying for up to five
days. 15

Clearly, the potential value of sugars, such as trehalose and sucrose, has been recognized for
many years. We have been working on the metal-actuated switchable membrane pore system to
reversibly permeabilize mammalian cells to sugars. In the course of these studies, short-term
preincubation with sugars, prior to poration, resulted in improved experimental outcomes.
These observations led to the testing of longer incubation times without subsequent poration as
controls for possible sugar effects. These experiments led to the unanticipated discovery that
incubation of cellular materials under physiological conditions in the presence of low
concentrations of sugars resulted in increased cell survival without any need for the metal-
actuated switchable membrane pore.

Definitions: Preservation - a process for provision of shelf life to a cell containing, living
biological material. Preservation processes include cryopreservation by freezing and vitrification
and anhydrobiotic preservation by either freeze-drying or dessication. Living biological
materials includes all materials natural or man made with cellular components.

Summary of the invention: The present application provides a method for pre-treatment of
cellular materials with sugars that enhances the ability of said cellular materials to survive a
subsequent preservation procedure. Incubation of cellular materials in sugars under physiological
conditions for short periods of time (less than 3 hours), with or without simple addition of
extracellular sugars just prior to cell preservation, results in few, if any, cells surviving
preservation procedures. However, we have discovered that prolonged incubation with sugars
under physiological conditions (greater than 3 hours) prior to preservation results in cell survival
under conditions that would otherwise have resulted in minimal, if any, cell survival.

Method: A bovine pulmonary artery endothelial cell line, CPAE, was used for these
experiments. Cells were plated the night before in 96-well microtiter plates at 20,000 cells/well,
then exposed to 0.2M trehalose in Dulbecco's Modified Eagle's Medium (DMEM) at 37°C for 0,
3, 6, 12, 18, 24, 48 and 72 hours. Following exposure and prior to freezing, cell viability was
determined using the non-invasive metabolic indicator alamarBlue (Trek Diagnostics). A volume
of 20 ul was added to cells in 200 ul of DMEM(10%FCS) and the plate was allowed to incubate
at 37 °C for three hours. The plate was read using a fluorescent microplate reader (Molecular
Dynamics) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm. The
alamarBlue assay was chosen because it is non-toxic and can be used to assess the viability of
the cells without damage. Thus, we could check cell viability and then immediately freeze the
cells in the plate. Cells were subsequently placed in 0-1. 0M trehalose in DMEM (50 ul) and
immediately cryopreserved using a controlled-rate freezer at -1.0°C/min. The following day, the
cells were thawed by incubation for 30 minutes at -20°C, followed by rapid thawing at 37°C.
The trehalose was diluted with 150 ul of DMEM (10% fetal calf serum, FCS) and the cells were
left for one hour at 37°C. Cell viability was then reassessed using alamarBlue by incubation for
three hours. Cell viability was also assessed on day four or five post-thaw.

Claims: Our first reaction is that the claims should focus on "A method for treatment of living
biological materials (cells, tissues and organs) that enhances (increases, improves?) survival of
cells that are subsequently subjected to preservation protocols." Essentially we have discovered
that periods of incubation under tissue culture conditions in the presence of sugars
(disaccharides, trehalose effect demonstrated to date) for periods greater than three hours results
in enhanced post-preservation cell survival.

In our first experiments we have used 0.2 M trehalose incubation with 0.2-1.0 M of extracellular
trehalose being added at the time of preservation, all extracellular concentrations provide cell
survival benefits. Neither incubation alone nor extracellular sugars alone conferred any cell
survival.

Experiments are in progress to assess some other opportunities including:

1) We anticipate that addition of other cryoprotectants either with or without extracellular
sugars will result in enhanced cell survival. We will want to incorporate the list of
reagents we have used in prior cryopreservation patents in a dependant claim.

2) It is also possible that modification of culture conditions may result in further
improvements in cell survival post-sugar incubation.

3) It is possible that other sugar concentrations or nutrient conditions may result in positive
effects after less than three hours of incubation.

REFERENCES CITED

1 . Polge C, A.Y. Smith & A.S. Parkes. 1949. Revival of spermatozoa after vitrification and
de-hydration at low temperatures. Nature, 164:666.

2. Lovelock J.E. & M.W.H. Bishop. 1 959. Prevention of freezing damage to living cells by
dimethylsulphoxide. Nature, 183:1394-1395.

3. Brockbank K.G.M. & K.M. Smith. 1993. Synergistic Interaction Of Low-Molecular
Weight Polyvinyl-pyrrolidones with Dimethylsulfoxide during Cell Cryopreservation.
Transplant Proc. 25:3 1 85-3 1 97.

4. Song Y.C. et al. , 2000. Vitreous cryopreservation maintains the function of vascular
grafts. Nature Biotechnology, 18:296 -299.

5. Crowe J.H. et al. , 1 998. The role of vitrification in anhydrobiosis. Ann Rev Physiol. 60:
73 - 103.

6. Potts M. 1994. Desiccation tolerance of prokaryotes. Microbiol Rev 58: 755 - 805.

7. Crowe J.H. et al. , 1 988. Interactions of sugars with membranes. Biochim Biophys Acta
947:367- 384.

8. Crowe J.H. et al, 1993. Anhydrobiosis. Ann Rev Physiol, 54:579-599.

9. Bieganski R.M. et al, 1998. Stabilization of active recombinant retroviruses in an
amorphous dry state with trehalose. Biotechnol Prog 14:615- 620.

10. Womersley C. et al, 1986. Inhibition of dehydration-induced fusion between liposomal
membranes by carbohydrates as measured by fluorescence energy-transfer. Cryobiology
23:245 - 255.

11. Castro, A.G., Lapinski, J., Tunnacliffe, A. Nature Biotechnology 18:473, 2000.

12. Eroglu A et al, 2000. Intracellular trehalose improves the survival of cryopreserved
mammalian cells. Nature Biotechnology 18:163 - 167. U.S Patent for preservation of
biological Materials allowed, 2001.

13. Beattie, G.M., Crowe, J.H., Tablin, F., Hayek, A. Cryopreservation of human adult and
fetal pancreatic cells and human platelets. US Patent #5,827,741, 1998.

14. Beattie, G.M., Crowe, J.H., Lopez, A. D., Cirulli, V., Ricordi, C, Hayek, A. Trehalose: a
cryoprotectant that enhances recovery and preserves function of human pancreatic islets
after long-term storage. Diabetes 46:519-523, 1997.

15. Acker, J.P., Toner, M. Tissue Engineering: Biopreservation. Science and Medicine, 126-
127, 2002.